外源性IL-4通过抑制NF-κB信号通路减轻急性脓毒症肺损伤的研究

Exogenous IL-4 attenuates lung injury in acute sepsis by inhibiting NF-κB signaling pathway

目的通过构建脓毒症肺损伤体内及体外模型,探讨外源性白细胞介素-4(IL-4)可否减轻脓毒症急性肺损伤及其可能的调节机制。方法细胞实验:体外培养小鼠肺泡巨噬细胞MH-S,选用终浓度为1.0μg/mL的脂多糖(LPS)刺激2 h后,加入不同浓度的IL-4作用24 h,CCK-8法检测细胞活力;活性氧(ROS)测定试剂盒检测ROS的含量,Elish法检测IL-1β、IL-6、TNF-α表达水平,Western blot法检测NF-κB p65蛋白表达水平。小鼠实验:选用C57BL/6小鼠,随机分为空白对照组(生理盐水)、模型组(LPS)及实验组(LPS造模30 min后气管滴注IL-4),12 h后,将3组小鼠经异氟醚诱导后处死收集血清和组织样本。使用H&E染色法观察肺损伤,采用qRT-PCR和ELISA法检测相关基因的表达,流式细胞术检测巨噬细胞的极化情况。结果外源性IL-4可明显改善肺损伤。体外实验表明,外源性IL-4在24 h内对MH-S细胞的活力几乎无影响;外源性IL-4能够降低MH-S细胞中TNF-α、IL-6、IL-1β水平,下调NF-κB p65蛋白表达量以及减少ROS的表达(P<0.05)。体内实验表明,外源性IL-4通过下调相关基因的表达增加了F4/80+CD206+M2样巨噬细胞,抑制了F4/80+iNOS+M1样巨噬细胞的分布(P<0.05)。此外,外源性IL-4可以影响小鼠iNOS、TNF-α、MCP-1、IL-10的蛋白表达,同时LPS诱导的p65水平升高也被外源性IL-4所抑制。结论外源性IL-4能够抑制促炎细胞因子的表达、减少ROS生成、降低肺组织病理评分和W/D值,对改善肺损伤有积极作用。此外,外源性IL-4可促进巨噬细胞极化,并抑制NF-κB/p65信号通路激活。

Objective To explore whether exogenous IL-4 can attenuate acute lung injury in sepsis by constructing models of sepsis lung injury in vivo and in vitro,and explore its possible regulatory mechanisms.Methods MH-S mouse alveolar macrophages were cultured in vitro and stimulated by lipopolysaccharide(LPS)at a final concentration of 1.0μg/mL for 2 h.The different concentrations of IL-4 was added for 24 h.Cell viability was measured by CCK-8 assay.The content of reactive oxygen species(ROS)was measured by ROS assay kit.IL-1β,IL-6 and TNF-α were measured by Elisa assay.The expression levels of IL-1β,IL-6 and TNF-α were measured by the ELISA.The expression of NF-κB p65 protein were measured by Western blot.C57BL/6 mice were randomly divided into control group(saline),model group(LPS)and experimental group(IL-4,was injected into the trachea 30 min after LPS modeling).After 12 hours,the mice in the three groups were induced by isoflurane and then executed to collect serum and tissue samples.The lung injury was observed by using H&E staining,and the expression of related genes was detected by qRT-PCR and ELISA,and the polarization of macrophages was detected by flow cytometry.Results Exogenous IL-4 significantly improved lung injury.In vitro,exogenous IL-4 had little effect on the viability of MH-S cells within 24 h.Exogenous IL-4 was able to reduce the levels of TNF-α,IL-6,IL-1β in MH-S cells,and down-regulate the expression of NF-κB p65 protein,as well as reduce the content of ROS in MH-S cells(P<0.05).In vivo,exogenous IL-4 increased F4/80+CD206+M2-like macrophages and inhibited the distribution of F4/80+iNOS+M1-like macrophages by down-regulating the expression of related genes(P<0.05).In addition,exogenous IL-4 could affect the protein expression of iNOS,TNF-α,MCP-1,and IL-10 in mice.Meanwhile,the increase of p65 level induced by LPS was also inhibited by exogenous IL-4.Conclusion Exogenous IL-4 can inhibit the expression of pro-inflammatory cytokines,reduce the production of ROS,the lung histopathological scores and W/D values.Exogenous IL-4 have a positive effect on improving lung injury.In addition,exogenous IL-4 can promote macrophage polarization and inhibite the activation of NF-κB/p65 signaling pathway.